CN108103149A - Detection Nucleic acid combinations, kit, chip and its application of a kind of Fusobacterium nucleatum - Google Patents
Detection Nucleic acid combinations, kit, chip and its application of a kind of Fusobacterium nucleatum Download PDFInfo
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- CN108103149A CN108103149A CN201810174817.9A CN201810174817A CN108103149A CN 108103149 A CN108103149 A CN 108103149A CN 201810174817 A CN201810174817 A CN 201810174817A CN 108103149 A CN108103149 A CN 108103149A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The present invention provides detection Nucleic acid combinations, kit, chip and its applications of a kind of Fusobacterium nucleatum, belong to bioassay quarantine field.The detection Nucleic acid combinations of Fusobacterium nucleatum provided by the invention can exactly, specifically detect Fusobacterium nucleatum, pass through the use of multiple bonding probes, the accuracy and accuracy of detection of raising detection that can be larger, beneficial to obtaining accurate testing result, it can preferably avoid the problem that false positive and false negative, the detection Nucleic acid combinations of Fusobacterium nucleatum are applied in the detection kit for preparing Fusobacterium nucleatum detection, detection chip, it is obtained to check that kit, detection chip obtain accurate testing result, there is preferable actual application value.
Description
Technical field
It quarantines field the present invention relates to bioassay, a kind of detection Nucleic acid combinations in particular to Fusobacterium nucleatum,
Kit, chip and its application.
Background technology
Fusobacterium nucleatum (Fusobacterium nucleatum) belongs to Gram-negative obligate anaerobe, participates in periodontal
The inflammatory reaction of disease is one of putative pathogen of periodontitis.The bacterium, can also be in septicemia phase in addition to it can cause mouth disease
It separates and obtains in the abscess and Colorectal Carcinoma of the internal organs such as pass infection, pelvic infecton, brain, liver, lung, spleen.Recently on tool core shuttle
Bacillus and the research of pre-term low birth weight, colorectal cancer and respiratory tract infection correlation become hot spot.There are the studies have shown that mankind big
The surface enrichment of intestinal cancer, metastatic tumor and preclinical colorectal cancer cells has a kind of sugar for being known as Gal-GalNA, Fusobacterium nucleatum surface
Fap2 albumen can be combined with Gal-GalNAc.When the Fusobacterium nucleatum for expressing Fap2 enters colon by blood circulation, just
It can colonize on colon tumor in the original location, and be proliferated in tumor locus, so as to accelerate the development of colorectal cancer；Also by inducing cancer cell
Autophagy and the postoperative recurrence mechanism for causing chemotherapy resistance and tumour, so that PATIENTS WITH LARGE BOWEL five year survival rate reduces.Therefore,
Rapid identification and detection to Fusobacterium nucleatum will provide for systemic diseases such as prevention and treatment oral cavity, premature labor, straight colon cancers
New thinking.
At present, the detection of Fusobacterium nucleatum mainly has Bacteria Culture and identification technology, nucleic acid probe hybridization technology and is immunized
Tissue chemical technology.The culture of bacterium and identification technology are most basic reliable methods and micro- in traditional microorganism detection
The goldstandard that biology checks, other detection techniques are frequently necessary to compared with it, so its also clinically extensive use.
Since the condition that anaerobic bacteria is separately cultured is more demanding, the time is longer, and clinical samples taken amount is few, relevant bacteria species
It is more, so clinical being separately cultured for use is subjected to limitation with identification method.Full genome probe may be deposited with bacterium not of the same race
In cross reaction, cross reaction may occur between homologous bacterial for the DNA probe of clone, the vacation sun that anaerobic bacteria is caused to detect
Property result.For example, cross reaction, and any one may occur with Escherichia coli and Klebsiella pneumoniae etc. for Fusobacterium nucleatum
Kind method can not possibly have 100% specificity and sensibility, so nucleic acid probe hybridization technology is susceptible to false positive and vacation is cloudy
The problem of property.Immunofluorescence and enzyme linked immunosorbent assay (ELISA) need to prepare antibody, and process is cumbersome, and time-consuming, though and its stability it is good
But poor specificity.
The content of the invention
The first object of the present invention is the detection Nucleic acid combinations for providing a kind of Fusobacterium nucleatum, the detection Nucleic acid combinations energy
Sensitively, it is specific react, Fusobacterium nucleatum is accurately tested and analyzed.
The second object of the present invention is that the detection Nucleic acid combinations for providing above-mentioned Fusobacterium nucleatum are examined in Fusobacterium nucleatum
Application in survey.
The third object of the present invention is that the detection Nucleic acid combinations for providing above-mentioned Fusobacterium nucleatum are preparing tool core shuttle bar
Application in the detection kit of bacterium.
The fourth object of the present invention is that the detection Nucleic acid combinations for providing above-mentioned Fusobacterium nucleatum are preparing tool core shuttle bar
Application in the detection chip of bacterium.
The fifth object of the present invention is to provide a kind of detection kit of Fusobacterium nucleatum, which can facilitate
Quickly it is applied in the detection of Fusobacterium nucleatum.
The sixth object of the present invention is to provide a kind of detection chip of Fusobacterium nucleatum, can be answered by the detection chip
In the detection for using great amount of samples, quickly detection is realized.
The seventh object of the present invention is to provide the application that above-mentioned detection chip is detected in Fusobacterium nucleatum.
The eigth object of the present invention is to provide a kind of preparation method of the detection chip of Fusobacterium nucleatum, by preparation side
Method prepares the chip detected for Fusobacterium nucleatum by simply putting step.
In order to realize the above-mentioned purpose of the present invention, using following technical scheme：
A kind of detection Nucleic acid combinations of Fusobacterium nucleatum, detection Nucleic acid combinations bag detection nucleic acid and internal reference nucleic acid；
Detecting nucleic acid includes detection primer pair and detection probe；The base sequence of detection primer pair such as SEQ ID NO.1-2
It is shown；Detection probe is the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combination
At least one of probe, the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combine
The base sequence of probe is as shown in SEQ ID NO.3-7；
Internal reference nucleic acid includes internal control primer pair and internal reference probe, the base sequence such as SEQ ID NO.8-9 of internal control primer pair
Shown, the base sequence of internal reference probe is as shown in SEQ ID NO.10；
And negative control probe, the base sequence of negative control probe is as shown in SEQ ID NO.11.
The template sequence of internal reference is TCTCTTAGATATGCAATAATTTTCCCACTATGACTGAGTCACGTACTCCACAT
TTTCAAATTTGTGTCTTCTGTTC。
Application of the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum in Fusobacterium nucleatum detects.
Application of the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum in the detection kit for preparing Fusobacterium nucleatum.
Application of the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum in the detection chip for preparing Fusobacterium nucleatum.
A kind of detection kit of Fusobacterium nucleatum, detection kit include the detection nucleic acid group of above-mentioned Fusobacterium nucleatum
It closes.
A kind of detection chip of Fusobacterium nucleatum, detection chip are included in the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum
Detection probe, internal reference probe and negative control probe.
The application that above-mentioned detection chip is detected in Fusobacterium nucleatum.
A kind of preparation method of the detection chip of Fusobacterium nucleatum, including：
With amino acid modification glass substrate, by the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum successively point sample 0.1-0.3 μ
L is incubated 1-3h under 75-88 DEG C of temperature conditionss, and the detection chip of Fusobacterium nucleatum is made.
Compared with prior art, beneficial effects of the present invention are：The detection nucleic acid group of Fusobacterium nucleatum provided by the invention
Close can exactly, specifically detect Fusobacterium nucleatum, by the use of multiple bonding probes, raising detection that can be larger it is accurate
Property and accuracy of detection, beneficial to accurate testing result is obtained, can preferably avoid the problem that false positive and false negative, will have core shuttle
The detection Nucleic acid combinations of bacillus are applied in the detection kit for preparing Fusobacterium nucleatum detection, detection chip, inspection obtained
Kit, detection chip can obtain accurate testing result, have preferable actual application value.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Below to detection Nucleic acid combinations, kit, chip and its application of a kind of Fusobacterium nucleatum of the embodiment of the present invention
It is specifically described.
A kind of detection Nucleic acid combinations of Fusobacterium nucleatum, detection Nucleic acid combinations bag detection nucleic acid and internal reference nucleic acid；
Detecting nucleic acid includes detection primer pair and detection probe；The base sequence of detection primer pair such as SEQ ID NO.1-2
It is shown；Detection probe is the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combination
At least one of probe, the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combine
The base sequence of probe is as shown in SEQ ID NO.3-7；
Internal reference nucleic acid includes internal control primer pair and internal reference probe, the base sequence such as SEQ ID NO.8-9 of internal control primer pair
Shown, the base sequence of internal reference probe is as shown in SEQ ID NO.10；
And negative control probe, the base sequence of negative control probe is as shown in SEQ ID NO.11.
Further, in presently preferred embodiments of the present invention, 5 ' ends of the sense primer of detection primer pair include the first fluorescence
Group, 5 ' ends of the anti-sense primer of detection primer pair include the second fluorophor, 5 ' end bags of the sense primer of internal control primer pair
Include the 3rd fluorophor；First fluorophor for FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610,
One kind in TEXASRED, RED670 or NED；Second fluorophor for FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5,
One kind in ROX, RED610, TEXASRED, RED670 or NED；3rd fluorophor for FAM, MGB, VIC, TET, JOE,
One kind in HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 or NED；First fluorophor, the second fluorophor
It is differed with the 3rd fluorophor.
Select a kind of fluorophor when the first fluorophor, the second fluorophor and the 3rd fluorophor difference, it is convenient into
Row fluorescence is distinguished, and is more advantageous to the progress of detection.
Application of the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum in Fusobacterium nucleatum detects.
Application of the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum in the detection kit for preparing Fusobacterium nucleatum.
Application of the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum in the detection chip for preparing Fusobacterium nucleatum.
A kind of detection kit of Fusobacterium nucleatum, detection kit include the detection nucleic acid group of above-mentioned Fusobacterium nucleatum
It closes.
Further, in presently preferred embodiments of the present invention, Premix Taq are further included.
A kind of detection chip of Fusobacterium nucleatum, detection chip are included in the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum
Detection probe, internal reference probe and negative control probe.
The application that above-mentioned detection chip is detected in Fusobacterium nucleatum.
A kind of preparation method of the detection chip of Fusobacterium nucleatum, including：
With amino acid modification glass substrate, by the detection Nucleic acid combinations of above-mentioned Fusobacterium nucleatum successively point sample 0.1-0.3 μ
L is incubated 1-3h under 75-88 DEG C of temperature conditionss, and the detection chip of Fusobacterium nucleatum is made.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides the detection Nucleic acid combinations of Fusobacterium nucleatum, detection Nucleic acid combinations bag detection nucleic acid and internal reference core
Acid；
Detecting nucleic acid includes detection primer pair and detection probe；The base sequence of detection primer pair such as SEQ ID NO.1-2
It is shown；Detection probe is the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combination
At least one of probe, the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combine
The base sequence of probe is as shown in SEQ ID NO.3-7；
Internal reference nucleic acid includes internal control primer pair and internal reference probe, the base sequence such as SEQ ID NO.8-9 of internal control primer pair
Shown, the base sequence of internal reference probe is as shown in SEQ ID NO.10；
And negative control probe, the base sequence of negative control probe is as shown in SEQ ID NO.11.
5 ' ends of the sense primer of detection primer pair include the first fluorophor, 5 ' ends of the anti-sense primer of detection primer pair
Including the second fluorophor, 5 ' ends of the sense primer of internal control primer pair include the 3rd fluorophor；First fluorophor is selected
MGB；Second fluorophor selects TET；3rd fluorophor selects ROX.
Certainly, the first fluorophor can also be MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610,
One kind in TEXASRED, RED670 or NED；Second fluorophor can also be MGB, VIC, TET, JOE, HEX, CY3, CY5,
One kind in ROX, RED610, TEXASRED, RED670 or NED；3rd fluorophor can also be MGB, VIC, TET, JOE,
One kind in HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 or NED；And first fluorophor, the second fluorescent base
Identical fluorophor is selected when group and the 3rd fluorophor different between any two.
Therefore, the detection Nucleic acid combinations of Fusobacterium nucleatum provided in this embodiment can be applied to detection Fusobacterium nucleatum
In, the detection kit and detection chip that prepare Fusobacterium nucleatum can also be applied to.
Embodiment 2
The present embodiment provides the detection Nucleic acid combinations of Fusobacterium nucleatum, detection Nucleic acid combinations bag detection nucleic acid and internal reference core
Acid；
Detecting nucleic acid includes detection primer pair and detection probe；The base sequence of detection primer pair such as SEQ ID NO.1-2
It is shown；Detection probe is the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combination
At least one of probe, the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combine
The base sequence of probe is as shown in SEQ ID NO.3-7；
Internal reference nucleic acid includes internal control primer pair and internal reference probe, the base sequence such as SEQ ID NO.8-9 of internal control primer pair
Shown, the base sequence of internal reference probe is as shown in SEQ ID NO.10；
And negative control probe, the base sequence of negative control probe is as shown in SEQ ID NO.11.
5 ' ends of the sense primer of detection primer pair include the first fluorophor, 5 ' ends of the anti-sense primer of detection primer pair
Including the second fluorophor, 5 ' ends of the sense primer of internal control primer pair include the 3rd fluorophor；First fluorophor is selected
FAM；Second fluorophor selects VIC；3rd fluorophor selects CY3.
Embodiment 3
The present embodiment provides a kind of detection kit of Fusobacterium nucleatum, which is included for Fusobacterium nucleatum
Detect Nucleic acid combinations, detection Nucleic acid combinations bag detection nucleic acid and internal reference nucleic acid；
Detecting nucleic acid includes detection primer pair and detection probe；The base sequence of detection primer pair such as SEQ ID NO.1-2
It is shown；Detection probe is the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combination
At least one of probe, the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes or the 5th combine
The base sequence of probe is as shown in SEQ ID NO.3-7；
Internal reference nucleic acid includes internal control primer pair and internal reference probe, the base sequence such as SEQ ID NO.8-9 of internal control primer pair
Shown, the base sequence of internal reference probe is as shown in SEQ ID NO.10；
And negative control probe, the base sequence of negative control probe is as shown in SEQ ID NO.11.
5 ' ends of the sense primer of detection primer pair include the first fluorophor, 5 ' ends of the anti-sense primer of detection primer pair
Including the second fluorophor, 5 ' ends of the sense primer of internal control primer pair include the 3rd fluorophor；First fluorophor is selected
FAM；Second fluorophor selects VIC；3rd fluorophor selects CY3.
Further include the reaction solution Premix Taq for carrying out PCR.
The reaction system for carrying out PCR is as shown in table 1.
Table 1PCR reaction systems
| Premix Taq | 5μL |
| Detection primer (upstream) (10 μM) | 0.2μL |
| Detection primer (downstream) (10 μM) | 0.2μL |
| Expand template (50-150ng/ μ L) | 0.4μL |
| Internal control primer (upstream) (10 μM) | 0.2μL |
| Interior label primer (downstream) (10 μM) | 0.2μL |
| Internal standard template (50ng/ μ L) | 0.4μL |
| Distilled water | 3.4μL |
The program of PCR reactions is 95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 30
A cycling；72 DEG C, 10min.
Embodiment 4
The present embodiment provides a kind of detection chip of Fusobacterium nucleatum, which includes the tool core shuttle in embodiment 1
Detection probe, internal reference probe and negative control probe in the detection Nucleic acid combinations of bacillus.
The preparation method of the detection chip of above-mentioned Fusobacterium nucleatum includes step in detail below:
1.1 pairs of glass substrate amino acid modifications；
1.2 arrange according to the dot matrix shown in table 2, deposition probe；
1.3 30 μM of concentration and probe concentrations, it is each to put 0.2 μ L of point sample；
1.4 are incubated 3h under the conditions of 75 DEG C, and the detection chip of Fusobacterium nucleatum is made.
2 probe arrangement model of table
Embodiment 5
The present embodiment provides a kind of detection chip of Fusobacterium nucleatum, which includes the tool core shuttle in embodiment 1
Detection probe, internal reference probe and negative control probe in the detection Nucleic acid combinations of bacillus.
The preparation method of the detection chip of above-mentioned Fusobacterium nucleatum includes step in detail below:
1.1 pairs of glass substrate amino acid modifications；
1.2 arrange according to the dot matrix shown in table 2, deposition probe；
1.3 30 μM of concentration and probe concentrations, it is each to put 0.2 μ L of point sample；
1.4 are incubated 1h under the conditions of 85 DEG C, and the detection chip of Fusobacterium nucleatum is made.
Embodiment 6
The present embodiment provides a kind of detection chip of Fusobacterium nucleatum, which includes the tool core shuttle in embodiment 1
Detection probe, internal reference probe and negative control probe in the detection Nucleic acid combinations of bacillus.
The preparation method of the detection chip of above-mentioned Fusobacterium nucleatum includes step in detail below:
1.1 pairs of glass substrate amino acid modifications；
1.2 arrange according to the dot matrix shown in table 2, deposition probe；
1.3 30 μM of concentration and probe concentrations, it is each to put 0.2 μ L of point sample；
1.4 are incubated 1.5h under the conditions of 80 DEG C, and the detection chip of Fusobacterium nucleatum is made.
Experimental example 1
This experimental example utilizes the detection kit of Fusobacterium nucleatum of the offer of embodiment 3 and the Fusobacterium nucleatum of embodiment 6
Detection chip be detected.
On the genetic chip prepared, by the pcr amplification product point sample of sample to be tested to the first bonding probes, the second knot
In the detection hole for closing probe, the 3rd bonding probes, the 4th bonding probes and the 5th bonding probes, meanwhile, by positive control
It is corresponding to add in positive control and blank control detection hole with blank control sample PCR product.
It concretely comprises the following steps:The unwinding 5 minutes of 95 DEG C of a.PCR products, is immediately placed on ice, PCR product and hybridization buffer
(50% formamide, 10XSSC, 0.2%SDS) is mixed in equal volume, and 10 microlitres of mixed liquors are selected per hole, in wet box, in 60 DEG C of hybridization
Be incubated 2 it is small when.B. the genetic chip after being incubated is taken out, is cleaned 3 times with cleaning solution (0.3XSSC buffer solutions).C. chip is put into
Laser co-focusing detector (GenePix) scans chip at 650nm:Software analysis of fluorescence signal strength values, provide result.
In conclusion the detection Nucleic acid combinations of Fusobacterium nucleatum provided in an embodiment of the present invention are joined by multiple bonding probes
It closes and uses, the precision and sensitivity of detection can be improved, can significantly avoid the problem that false positive and false negative so that detection knot
Fruit is more accurate, and the detection Nucleic acid combinations of Fusobacterium nucleatum are applied to the detection kit and detection chip for preparing Fusobacterium nucleatum
In, the detection kit and detection chip that are prepared are likewise supplied with high accuracy and sensitivity, improve the standard of testing result
True property has preferably actual application value.
Embodiments described above is part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
Every other embodiment, belongs to the scope of protection of the invention.
SEQUENCE LISTING
<110>Sichuan Zhi Ji future healths Management Co., Ltd
<120>Detection Nucleic acid combinations, kit, chip and its application of a kind of Fusobacterium nucleatum
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
caactctgta ttgcgttgga actgt 25
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
gcgtcgaatt aaaccacatg c 21
<210> 3
<211> 37
<212> DNA
<213>Artificial sequence
<400> 3
tttttttttt ctagagtact ggagaggtaa gcggaac 37
<210> 4
<211> 30
<212> DNA
<213>Artificial sequence
<400> 4
tttttttttt ccgatgggga agccagctta 30
<210> 5
<211> 30
<212> DNA
<213>Artificial sequence
<400> 5
tttttttttt ctcagcgccc aagcaaaacg 30
<210> 6
<211> 38
<212> DNA
<213>Artificial sequence
<400> 6
tttttttttt aattcctttg agtttcatac ttgcgtac 38
<210> 7
<211> 31
<212> DNA
<213>Artificial sequence
<400> 7
tttttttttt cgtactcccc aggcggatta c 31
<210> 8
<211> 31
<212> DNA
<213>Artificial sequence
<400> 8
tctcttagat atgcaataat tttcccacta t 31
<210> 9
<211> 23
<212> DNA
<213>Artificial sequence
<400> 9
tttcaaattt gtgtcttctg ttc 23
<210> 10
<211> 32
<212> DNA
<213>Artificial sequence
<400> 10
tttttttttt gactgagtca cgtactccac at 32
<210> 11
<211> 33
<212> DNA
<213>Artificial sequence
<400> 11
tttttttttt gactgagtca cgtactccac aat 33
Claims (10)
- A kind of 1. detection Nucleic acid combinations of Fusobacterium nucleatum, which is characterized in that the detection Nucleic acid combinations bag detection nucleic acid and interior Join nucleic acid；The detection nucleic acid includes detection primer pair and detection probe；The base sequence of the detection primer pair such as SEQ ID Shown in NO.1-2；The detection probe is the first bonding probes, the second bonding probes, the 3rd bonding probes, the 4th bonding probes Or the 5th at least one of bonding probes, first bonding probes, second bonding probes, the 3rd bonding probes, The base sequence of 4th bonding probes or the 5th bonding probes is as shown in SEQ ID NO.3-7；The internal reference nucleic acid includes internal control primer pair and internal reference probe, the base sequence such as SEQ ID of the internal control primer pair Shown in NO.8-9, the base sequence of the internal reference probe is as shown in SEQ ID NO.10；And negative control probe, the base sequence of the negative control probe is as shown in SEQ ID NO.11.
- 2. the detection Nucleic acid combinations of Fusobacterium nucleatum according to claim 1, which is characterized in that the detection primer pair 5 ' ends of sense primer include the first fluorophor, and 5 ' ends of the anti-sense primer of the detection primer pair include the second fluorescent base Group, 5 ' ends of the sense primer of the internal control primer pair include the 3rd fluorophor；First fluorophor for FAM, MGB, One kind in VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 or NED；Second fluorescence Group is one in FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 or NED Kind；3rd fluorophor for FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, One kind in RED670 or NED；First fluorophor, second fluorophor and the 3rd fluorophor not phase Together.
- 3. application of the detection Nucleic acid combinations of Fusobacterium nucleatum as claimed in claim 1 or 2 in Fusobacterium nucleatum detects.
- 4. the detection Nucleic acid combinations of Fusobacterium nucleatum as claimed in claim 1 or 2 are preparing the detection reagent of Fusobacterium nucleatum Application in box.
- 5. the detection Nucleic acid combinations of Fusobacterium nucleatum as claimed in claim 1 or 2 are preparing the detection chip of Fusobacterium nucleatum In application.
- 6. a kind of detection kit of Fusobacterium nucleatum, which is characterized in that the detection kit includes such as claim 1 or 2 The detection Nucleic acid combinations of the Fusobacterium nucleatum.
- 7. detection kit according to claim 6, which is characterized in that further include PremixTaq.
- 8. a kind of detection chip of Fusobacterium nucleatum, which is characterized in that the detection chip includes tool as described in claim 1 The detection probe, the internal reference probe and the negative control probe in the detection Nucleic acid combinations of core Fusobacterium.
- 9. the application that detection chip as claimed in claim 8 is detected in Fusobacterium nucleatum.
- 10. a kind of preparation method of the detection chip of Fusobacterium nucleatum, which is characterized in that including：With amino acid modification glass substrate, by the institute in the detection Nucleic acid combinations of Fusobacterium nucleatum as claimed in claim 1 or 2 Detection probe, the internal reference probe and the negative control probe point sample 0.1-0.3 μ L successively are stated, in 75-88 DEG C of temperature strip 1-3h is incubated under part, the detection chip of the Fusobacterium nucleatum is made.
Priority Applications (1)
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